Abstract

Elasmobranchs detect small potentials using excitable cells of the ampulla of Lorenzini which have calcium-activated K(+) channels, first described in 1974. A distinctive feature of the outward current in voltage clamped ampullae is its apparent insensitivity to voltage. The sequence of a BK channel ? isoform expressed in the ampulla of the skate was characterized. A signal peptide is present at the beginning of the gene. When compared to human isoform 1 (the canonical sequence), the largest difference was absence of a 59 amino acid region from the S8-S9 intra-cellular linker that contains the strex regulatory domain. The ampulla isoform was also compared with the isoform predicted in late skate embryos where strex was also absent. The BK voltage sensors were conserved in both skate isoforms. Differences between the skate and human BK channel included alternative splicing. Alternative splicing occurs at seven previously defined sites that are characteristic for BK channels in general and hair cells in particular. Skate BK sequences were highly similar to the Australian ghost shark and several other vertebrate species. Based on alignment of known BK sequences with the skate genome and transcriptome, there are at least two isoforms of Kcnma1? expressed in the skate. One of the ? subunits (?4), which is known to decrease voltage sensitivity, was also identified in the skate genome and transcriptome and in the ampulla. These studies advance our knowledge of BK channels and suggest further studies in the ampulla and other excitable tissues.

Abstract

Optical mapping is a tool used in cardiac electrophysiology to study the heart's normal rhythm and arrhythmias. The optical mapping technique provides a unique opportunity to obtain membrane potential recordings with a higher temporal and spatial resolution than electrical mapping. Additionally, it allows simultaneous recording of membrane potential and calcium transients in the whole heart. This article presents the basic concepts of optical mapping techniques as an introduction for students and investigators in experimental laboratories unfamiliar with it.

Abstract

Growth hormone (GH) is required to maintain normal cardiac structure and function and has a positive effect on cardiac remodeling in experimental and possibly human disease. Cardiac resistance to GH develops in the uremic state, perhaps predisposing to the characteristic cardiomyopathy associated with uremia. It was hypothesized that administration of low-dosage GH may have a salutary effect on the cardiac remodeling process in uremia, but because high levels of GH have adverse cardiac effects, administration of high-dosage GH may worsen uremic cardiomyopathy. In rats with chronic renal failure, quantitative cardiac morphology revealed a decrease in total capillary length and capillary length density and an increase in mean intercapillary distance and fibroblast volume density evident. Low-dosage GH prevented these changes. Collagen and TGF-beta immunostaining, increased in chronic renal failure, were also reduced by GH, suggesting a mechanism for its salutary action. Low-dosage GH also prevented thickening of the carotid artery but did not affect aortic pathology. In contrast, high-dosage GH worsened several of these variables. These results suggest that low-dosage GH may benefit the heart and possibly the carotid arteries in chronic renal failure.

Abstract

Alternation of cardiac action potential duration (APD) from beat to beat and concurrent alternation of the amplitude of the calcium transient are regarded as important arrhythmia mechanisms. These phenomena are causally interrelated and can be reliably evoked by an increase in beat frequency or by ischemia. The first part of this historical review deals with the physiology of APD alternans. Sections recounting the evolution of knowledge about calcium-activated ion currents and calcium transient alternans are interspersed among sections describing the growth of the so-called "restitution hypothesis," which involves time-dependent recovery of potassium channels (including their passage through pre-open states) as a function of diastolic interval. Major developments are generally in chronological order, but it is necessary to move back and forth between the two theories to respect the overall time line, which runs from about l965 to the present. The concluding two sections deal with the pathophysiology of calcium transient and APD alternans during ischemia, which may be the basis for out-of-hospital cardiac arrest during the initial stages of acute myocardial infarction.

Abstract

Cardiac ischemia causes beat-to-beat fluctuation in action potential duration (APD) alternans, which leads to T wave alternans and arrhythmias. Occurrence of APD alternans that is out of phase at two sites is especially important, but most APD alternans studies have involved rapid pacing of normal myocardium rather than ischemia. To determine the spatial features of APD alternans during ischemia, blood-perfused rabbit hearts were stained with 4-[beta-[2(di-n-butylamino)-6-napthyl]vinyl]pyridinium (di-4-ANEPPS) and imaged with a high-resolution camera. Hearts were perfused with oxygenated Tyrode solution at 37 degrees C for staining and then switched to a 50:50% blood/Tyrode mixture. Hearts were paced from the right ventricle at 3/s, and made ischemic by stopping flow for 6 min. Images of 10,000 pixels were obtained at 300 frames/s. Motion artifact was controlled by immobilization and by manual selection of undistorted single-pixel records. Upstroke propagation and conduction isochrones were displayed by computerized image processing. APD alternans was demonstrated in six of seven hearts, and was out of phase in different regions of the image in three hearts. The largest spatial variation in the onset of depolarization to 50% repolarization (APD50) was 155%. This caused beat-to-beat reversal of repolarization. An alternans map could be constructed for well-immobilized portions of the image. There were discrete regions of APD alternans separated by a boundary, as occurs with intracellular Ca2+ concentration alternans. Pixels as close together as 1.1 mm showed an APD alternans that was out of phase. The out-of-phase APD alternans was not due to conduction alternans, as shown by upstroke intervals and conduction isochrones. This contrasts with rapid pacing, where a causal relationship appears to exist. These new observations suggest distinct mechanisms for the genesis of arrhythmias during ischemia.

Abstract

Rapid progress has been made in understanding the molecular mechanisms by which calcium ions mediate certain cardiac arrhythmias. Principal advances include imaging of cytosolic calcium in isolated cells and in intact tissues, use of fluorescent indicators and monophasic action potentials to record membrane potentials in isolated tissue, and sequencing of the genes that encode critical ion channel proteins. In this review, five types of arrhythmias are discussed where calcium ion currents, or currents controlled by calcium, appear to be responsible for arrythmogenesis. These include: (1) the delayed afterpotential that occurs in conditions of intracellular calcium overload such as digitalis toxicity; (2) the early afterdepolarization that occurs when action potential duration is prolonged; (3) the slowly conducted calcium-dependent action potential (the slow response) in the SA and AV nodes; (4) the phenomenon of calcium transient alternans during ischemia, which is related to action potential duration alternans and t-wave alternans; (5) catecholamine-induced cardiac arrhythmias in families with mutations of the sarcoplasmic reticulum calcium-release channel. For each type of arrhythmia, the clinical implications of emerging knowledge are discussed. An especially important issue is whether ventricular fibrillation during acute coronary artery occlusion is due to calcium transient alternans. Ventricular fibrillation due to acute ischemia is an important subset of the 400,000 sudden cardiac deaths that occur annually in the U.S. Certain drugs, including beta blockers, fish oils, verapamil, and diltiazem, seem to specifically prevent ventricular fibrillation in this setting, and in most cases an effect of the drug on cytosolic calicum appears to be involved.

Abstract

Optical mapping of cytosolic calcium transients in intact mammalian hearts is now possible using long-wavelength [Ca(2+)](i) indicators. We propose that beat-to-beat [Ca(2+)](i) transient alternans during ischemia may lead to spatial and temporal heterogeneity of calcium-activated membrane currents.To test this hypothesis, isolated rabbit hearts were loaded with the fluorescent [Ca(2+)](i) indicator, rhod-2 AM, and imaged at 300 frames/sec during blood-perfused ischemic trials. High-quality [Ca(2+)](i) transients were recorded in each of 8 hearts.[Ca(2+)](i) transient alternans was never present in control records but occurred in each of the hearts during ischemia, with onset after 2 to 4 minutes. Alternans was confined to circumscribed regions of the heart surface 5 to 15 mm across. Multiple regions of alternans were found in most hearts, and regions that were out of phase with one another were found in 6 hearts. Quantitative maps of alternans were constructed by calculating an alternans ratio. This ratio behaved as a continuous variable that reached a maximum value in the center of the regions with alternans.These results demonstrate marked spatial heterogeneity of the [Ca(2+)](i) transient during the early phase of ischemia, which could produce electrical instability and arrhythmias in large mammalian hearts.

Abstract

Ischemia produces striking electrophysiological abnormalities in blood-perfused hearts that may be caused, in part, by effects of ischemia on intracellular calcium. To test this hypothesis, intracellular Ca2+ concentration ([Ca2+]i) transients were recorded from the epicardial surface of blood- and saline-perfused rabbit hearts using the long-wavelength indicator Fura Red. Calcium transients were much larger than the movement artifact, representing up to 29% of the total signal. Switching the perfusate from saline to blood did not affect the time course of the transients or the apparent level of [Ca2+]i. Compartmentation of Fura Red fluorescence was estimated by exposure to Mn2+. The results were cytosol 60 +/- 3%, organelles 12 +/- 2%, and autofluorescence plus partly deesterified Fura Red 29 +/- 4%. [Ca2+]i transients were calibrated in situ by perfusion of the extracellular space with high-Ca2+ and Ca(2+)-free EGTA solutions. Peak systolic [Ca2+]i was 663 +/- 74 nM, and end-diastolic [Ca2+]i was 279 +/- 59 nm. Ischemia was produced by interruption of aortic perfusion for 2.5 min during pacing (150 beats/min). Ischemia produced broadening of the [Ca2+]i transient, along with beat-to-beat alternations in the peak systolic and end-diastolic level of [Ca2+]i (calcium transient alternans). [Ca2+]i transient alternans occurred in 82% of blood-perfused hearts vs. 43% of saline-perfused hearts. The discrepancy between large and small transients (mean alternans ratio) was larger in the blood-perfused hearts (0.23 +/- 0.04 vs. 0.07 +/- 0.03, P = 0.005). These observations are important because of the apparent relationship of [Ca2+]i transient alternans to electrical alternans and arrhythmias during ischemia.

Abstract

1. Endothelin is a vasoactive peptide released from vascular endothelial cells which has potent cardiac inotropic effects. We examined the effect of endothelin on the verapamil-sensitive Ca2+ current (ICa) in enzymatically dispersed rabbit ventricular myocytes. 2. Using the whole-cell voltage clamp technique with a standard dialysing pipette solution, the application of extracellular endothelin (20 nM) did not increase the peak ICa, but in fact caused a small reversible decline (903 +/- 109 pA without endothelin, 727 +/- 95 pA with endothelin (means +/- S.E.M., n = 14, P less than 0.05)). 3. If GTP (100 microM) was added to the pipette solution, the extracellular application of endothelin (0.2 or 20 nM) caused a large, reproducible increase in peak ICa (871 +/- 85 pA without endothelin, 1230 +/- 110 pA with 20 nM-endothelin (n = 10, P less than 0.05). The endothelin enhancement of ICa occurred after a delay of approximately 3-4 min at room temperature. 4. The GTP requirement for the endothelin effect on ICa suggests that its effect may be mediated through a G protein-dependent pathway. To investigate this further, experiments were performed with pipette solutions containing guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), a GDP analogue which inhibits G protein cycling. With the addition of GDP beta S (0.5-5.0 mM) to the pipette solution (along with 100 microM-GTP), the effect of endothelin on peak ICa was blocked (1062 +/- 86 pA without endothelin, 1170 +/- 134 pA with endothelin (n = 11, P greater than 0.05)). 5. Incubation of myocytes with pertussis toxin (500 ng/ml) prevented the partial ACh-induced reversal of the isoprenolol enhancement of ICa. However, this identical treatment failed to block the endothelin enhancement of the voltage-dependent Ca2+ current (n = 4). 6. Taken together, these results confirm that while the effect of endothelin in rabbit cardiac ventricular myocytes is mediated through a G protein-dependent pathway, the G protein involved is pertussis toxin-insensitive.

Abstract

The time courses of changes in pHi and cytosolic calcium were compared in isolated perfused rabbit hearts with the use of the calcium-sensitive fluorescent indicator indo-1 and the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell-permeant forms of these indicators were loaded into myocytes by arterial infusion or by direct infusion into the extravascular space. Indo-1 fluorescence was recorded from the epicardial surface of the left ventricle at an excitation wavelength of 360 nm and emission wavelengths of 400 and 550 nm. BCECF fluorescence was recorded at an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Calibration procedures were developed for each indicator that allowed [Ca2+]i and pHi to be quantified during ischemia. Global ischemia decreased contractility and caused a rapid increase in both the systolic and end-diastolic levels of the calcium transients. Ninety seconds of ischemia increased peak systolic [Ca2+]i from 609 +/- 29 to 1,341 +/- 159 nM, while end-diastolic [Ca2+]i increased from 315 +/- 25 to 553 +/- 52 nM. The observed increase in diastolic [Ca2+]i, was shown not to arise from indo-1-loaded endothelial cells. The initial increase in [Ca2+]i was followed by a gradual decline and then a secondary rise occurring between 5 and 15 minutes of ischemia. In contrast, ischemia caused a monotonic decrease in pHi from a baseline of 7.03 +/- 0.06 to 6.83 +/- 0.02 after 2 minutes, 6.32 +/- 0.1 after 10 minutes, and 6.11 +/- 0.04 after 15 minutes. Perfusion of hearts with acidified (hypercarbic) saline increased the systolic and diastolic levels of the calcium transients, but only when pHi fell below a threshold value, which was more acidic than values achieved during the first 2 minutes of ischemia (6.83 +/- 0.03). Lesser degrees of acidification caused a decrease in contractility but did not affect the calcium transients. Effects of pHi on the calcium transients were not due to altered calcium sensitivity of indo-1. These results suggest that cytosolic acidification may contribute to the increase in [Ca2+]i during the first 15 minutes of global ischemia, but the [Ca2+]i increase during the first 2 minutes is mediated by other factors.

Abstract

The effect of platelet release products on cytosolic calcium [( Ca++]i) was examined by monitoring the fluorescence of chick embryonic heart cells loaded with the fluorescent calcium indicator indo-1 AM. Cell free filtrate of platelet release products was obtained from rabbit platelets activated with thrombin or collagen. This filtrate caused a rapid increase in both systolic and diastolic [Ca++]i in a dose-dependent manner. The effect was not blocked by pretreating the platelets with aspirin or a thromboxane synthetase inhibitor. It was not mimicked by a thromboxane analog, or by several substances known to be released from platelets including ADP, serotonin, or platelet activating factor. Apyrase or ATP-gamma S had no effect on the activity. The responsible product was heat-sensitive, trypsin-sensitive, and partitioned into the aqueous phase of a chloroform suspension. It has a low molecular weight (less than 3kD) and is sensitive to 2-mercaptoethanol. Protease inhibitor appears to prolong the activity. These results suggest that trypsin-sensitive peptide(s) released from activated platelets can increase [Ca++]i in cardiac cells.

Abstract

Thrombin increases intracellular calcium ([Ca++]i) in several cell types and causes a positive inotropic effect in the heart. We examined the mechanism of the thrombin-induced [Ca++]i increase in chick embryonic heart cells loaded with the fluorescent calcium indicator, indo-1. Thrombin (1 U/ml) increased both systolic and diastolic [Ca++]i from 617 +/- 62 and 324 +/- 46 to 1041 +/- 93 and 587 +/- 38 nM, respectively. An initial rapid [Ca++]i increase was followed by a more sustained increase. There were associated increases in contraction strength, beat frequency, and action potential duration. The [Ca++]i increase was not blocked by tetrodotoxin or verapamil, but was blocked by pretreatment with pertussis toxin (100 ng/ml). The thrombin-induced [Ca++]i increase was partly due to intracellular calcium release, since it persisted after removal of external calcium. The [Ca++]i increase in zero calcium was more transitory than in normal calcium and was potentiated by 10 mM Li+. Thrombin also induced influx of calcium across the surface membrane, which could be monitored using Mn++ ions, which quench indo-1 fluorescence when they enter the cell. Thrombin-induced Mn++ entry was insensitive to verapamil, but was blocked by 2 mM Ni++. Thrombin increased inositol trisphosphates by 180% at 90 s and this effect was also blocked by pretreatment with pertussis toxin. Conclusion: thrombin promotes calcium entry and release in embryonic heart cells even when action potentials are inhibited. Both modes of [Ca++]i increase may be coupled to the receptor by pertussis toxin-sensitive G proteins.

Abstract

Two of the major ionic abnormalities found early in ischemia are (a) loss of potassium with an increase in the extracellular potassium ion concentration and (b) an increase in free cytosolic calcium. Both of these ionic abnormalities can powerfully predispose to the development of arrhythmias.

Abstract

Single sodium channel openings have been recorded from cell-attached patches of isolated guinea pig ventricular myocytes. A paired pulse protocol was used to test the hypothesis that channel openings are required for lidocaine block. While the averaged ensemble current during the test pulse was much reduced, there was no correlation between the appearance of channel openings during the conditioning pulse and the subsequent test pulse. Analysis of single channel records demonstrated that the unit conductance of open channels was not changed by lidocaine. The block of ensemble INa was explained by roughly equal reductions in number of open channel events, and in the average duration of opening for each event. These results suggest that lidocaine binding to Na+ channels is dependent upon voltage, but may occur before channel opening. A lidocaine-modified channel can still open, but will be less likely to remain open than a drug-free channel. These results are consistent with block of a pre-open state of the channel.

Abstract

Cytosolic calcium transients were recorded from spontaneously beating chick embryonic myocardial cell aggregates loaded with the fluorescent [Ca2+]i indicator, indo-1. Calcium transients rose rapidly from an end-diastolic [Ca2+]i of 230 +/- 18 nM to a peak systolic [Ca2+]i of 619 +/- 34 nM (n = 21). Relaxation of the transients was slow, and continued throughout diastole. Bay K8644 (0.5 microM) markedly prolonged the action potential and caused similar prolongation of the calcium transients. Calcium transients in the presence of Bay K8644 had an inflection on their rising phase, which was followed by a more gradual increase that continued until the membrane had repolarized to a negative potential of -15 to -30 mV. Bay K8644 caused marked elevation of peak systolic [Ca2+]i to 955 +/- 56 nM (P less than 0.002), with concomitant elevation of end-diastolic [Ca2+]i to 400 +/- 36 nM (P less than 0.002). Optical recordings of contraction showed changes similar to those in the calcium transient: the initial upstroke of the contraction was followed by a more gradual second component, which gave the contraction a "half-dome" appearance. The time to peak [Ca2+]i and the time to peak contraction increased linearly with action potential duration (APD50). The effects of Bay K8644 were simulated, in part, by CsCl (7.5 mM), which produced equivalent prolongation of the action potential and calcium transients. However, CsCl did not elevate diastolic [Ca2+]i. To determine the mechanism of the diastolic [Ca2+]i increase, Bay K8644 was applied to aggregates rendered quiescent by tetrodotoxin. Bay K8644 caused a graded increase in [Ca2+]i, which was followed by resumption of spontaneous beating.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract

Although the Ca++ channel blockers can reduce early ischemic ventricular arrhythmias, the mechanisms are unclarified. The antiarrhythmic action of Ca++ antagonists may either be due to vasodilation and negative chronotropism or to trans-sarcolemmal Ca++ influx inhibition. In these studies we investigated the possible individual and additive effects of coronary flow, heart rate and Ca++ antagonism on ventricular arrythmia development in isolated, paced, globally underperfused guinea pig hearts. When the coronary flow during ischemia was raised from 5 to 7% of control and/or the stimulation frequency was decreased from 6 to 4 Hz, ATP and creatine phosphate levels were conserved and intraventricular conduction slowing leading to ventricular tachycardia (VT) was delayed. In contrast, when the coronary flow and pacing rates were fixed at 7% and 6 Hz and diltiazem (10(-6) M) was included in the perfusion medium, there was no effect on tissue high-energy phosphate depletion and development of VT. Even when the breakdown of ATP and the onset of VT were accelerated by isoprenaline (10(-6) M), diltiazem was not antiarrhythmic at this flow rate. Only when the coronary flow was reduced to 5% of control, in the absence and presence of isoprenaline, did diltiazem delay ventricular arrhythmias through a mechanism that was independent of changes in coronary flow and heart rate.

Abstract

The effects of acute global ischemia on cytosolic calcium transients were studied in perfused rabbit hearts loaded with the fluorescent calcium indicator indo 1. Indo 1-loaded hearts were illuminated at 360 nm, and fluorescence was recorded simultaneously at 400 and 550 nm from the epicardial surface of the left ventricle. The F400/F550 ratio was calculated by an analog circuit, which allowed cancellation of optical motion artifact. Resulting calcium transients demonstrated a rapid upstroke and slow decay similar to those recorded in isolated ventricular myocytes. Global ischemia rapidly suppressed contraction, but it produced a concurrent increase in the systolic and diastolic levels of the calcium transients, together with an increase in the duration of the peak. The effects of ischemia were reversed by reperfusion, inhibited by verapamil, and mimicked by perfusion of nonischemic hearts with acidified (CO2-rich) solution. In addition to elevation of the calcium transients, ischemia caused a pattern of intracellular calcium alternans that was discernible after 2-3 minutes. The pattern of alternans was stable at a given epicardial site, but it could be out of phase at different sites. Similar nonuniformities were observed in contraction strength and in the duration of monophasic action potentials recorded immediately adjacent to the fiber-optic probe. Abnormalities in intracellular calcium may be a causal factor in the loss of electrical and mechanical synchrony in the acutely ischemic heart.

Abstract

1. To study the origin of ischaemic myocardial depolarization, the diastolic surface potential - T-Q depression-was correlated with subepicardial extracellular K+ accumulation during serial episodes of widespread ischaemia in open-chest dogs, and in isolated, blood-perfused canine hearts. Placement of the reference electrode on a small island of non-ischaemic myocardium simplified the interpretation of the T-Q potentials. 2. In some experiments, changes in resting potential in the ischaemic zone were recorded using a 'contact' monophasic action potential (MAP) electrode. The change in MAP resting potential was linearly related to T-Q depression for a wide range of experimental conditions (R greater than 0.98). T-Q depression is therefore a linear index of depolarization in superficial myocardial cells. 3. The validity of T-Q depression as a 'measure' of local cellular depolarization was further tested by infiltration of isotonic KCl into the superficial myocardium subjacent to the ischaemic zone electrode. Resulting T-Q depression was 2- to 3-fold larger than the maximum values obtained in ischaemia; and the ratio of T-Q depression to the amplitude of the accompanying monophasic potential was consistent with the assumption that KCl had fully depolarized the underlying myocardium (delta Vm = 89 mV). KCl prevented (i.e. occluded) further changes in the T-Q potential during ischaemia. KCl did not have these effects if it was introduced at sites more remote from the electrode (greater than 4 mm). 4. Ischaemic T-Q depression was drastically accelerated by increasing the heart rate from 90 to 180 beats/min and was further accelerated by arterial infusion of CaCl2. These effects were most striking during the first minute of ischaemia. 5. In contrast, the above manoeuvres produced little acceleration of subepicardial K+ accumulation. After CaCl2 infusion, large ischaemic potentials, severe conduction impairment, and arrhythmias could be observed when K+ activity was almost normal (aK = 4.0-4.5 mM). 6. T-Q depression was larger in vivo than in isolated hearts, both absolutely and relative to K+ accumulation. 7. Based on the reproducible amplitude of ischaemic epicardial potential-estimates of cellular depolarization (delta Vm) could be obtained, which were compared with the concurrent change in K+ electrode potential (delta EK) for each experimental condition. 8. Estimated depolarization was nearly identical to delta EK in isolated hearts under basal conditions. However, depolarization significantly exceeded delta EK during rapid pacing, CaCl2 infusion, or during paced occlusions performed in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract

To elucidate the mechanism of the tension staircase, chick embryonic myocardial cell aggregates were loaded with the fluorescent cytosolic calcium indicator, indo 1. Fluctuations in indo 1 fluorescence were compared with recordings of cell edge movement during spontaneous beating and during stimulation by intracellular current pulses. Indo 1-loaded aggregates exhibit fluorescence transients during each transmembrane action potential. The rising phase of the transients is rapid, but the decaying phase is slow (several hundred msec) and is similar in time course to the pandiastolic relaxation seen in the optical recordings of cell edge movement. Acceleration of beat frequency by brief depolarizing current pulses produces an ascending staircase in both edge movement and systolic [Ca2+]i. There is a similar staircase in the diastolic [Ca2+]i, which is also reflected by diastolic cell edge movement. The existence of a diastolic [Ca2+]i staircase may provide new insight into the mechanism of the force-frequency relation in the heart.

CYTOSOLIC CALCIUM TRANSIENTS FROM THE BEATING MAMMALIAN HEARTPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALee, H. C., Smith, N., Mohabir, R., Clusin, W. T.1987; 84 (21): 7793-7797

Abstract

To elucidate the role of cytosolic calcium, [Ca2+]i, in the physiology of the normal and ischemic heart, we have developed a method for recording [Ca2+]i transients from the epicardial surface of the rabbit ventricle after arterial perfusion with the cell-permeant cytosolic calcium indicator indo-1 AM. Hearts were illuminated at 360 nm, and fluorescence was recorded simultaneously at 400 and 550 nm. The F400/F550 fluorescence ratio was calculated by an analog circuit that allowed cancelation of small movement artifacts that were present at single wavelengths. Clear [Ca2+]i transients were present in the F400/F550 signal and were remarkable for their slow decay. Slow decay of the transients was not due to buffering of [Ca2+]i by indo-1, since there was no associated impairment of contraction or relaxation. The peak amplitude of the [Ca2+]i transients was increased by ouabain, adrenaline, postextrasystolic potentiation, and acetylcholine. The extent to which the transients decayed diminished with shortening of the interbeat interval, but decay of the transients could be further diminished by acetylcholine or caffeine. A major advantage of the intact heart over isolated myocytes is the ability to measure changes in [Ca2+]i during ischemia. Ischemia produced a marked increase in both peak systolic and end-diastolic [Ca2+]i, which was most rapid during the first 30 sec, and approached a plateau value after 90 sec. This increase in [Ca2+]i was associated with a characteristic broadening of the peak of the transient. The increase in [Ca2+]i during ischemia is consistent with a proposed causative role of [Ca2+]i in mediating early electrophysiological abnormalities.

Abstract

Sarcolemmal sodium/calcium exchange activity was examined in individual chick embryonic myocardial cell aggregates that were loaded with quin 2. The baseline [Ca2+]i was 68 +/- 4 nM (n = 29). Abrupt superfusion with sodium-free lithium solution produced a fourfold increase in steady-state [Ca2+]i to 290 +/- 19 nM, which was reversible upon sodium restitution. Other methods of increasing [Ca2+]i such as KCl-depolarization or caffeine produced a dose-dependent increase in quin 2 fluorescence, accompanied by sustained contracture. The [Ca2+]i increase in zero sodium was linear, and its half-time (t1/2) of 15.1 +/- 0.1 s was similar to that of the sodium-free contracture (t1/2 = 14.4 +/- 0.5 s) under the same conditions. The sodium-dependent [Ca2+]i increase was not significantly greater when potassium served as the sodium substitute instead of lithium. This suggests that sodium/calcium exchange has little voltage dependence in this situation. However, in aggregates pretreated with ouabain (2.5 microM), the [Ca2+]i increase was almost threefold greater with potassium than with lithium (P less than 0.007). Ouabain therefore potentiated the effect of membrane potential on calcium influx. We propose that elevation of [Na2+]i is a prerequisite for voltage dependence of the sodium/calcium exchange under the conditions studied. Sodium loading will then drastically increase calcium influx during the action potential while inducing an outward membrane current that could accelerate repolarization.

Abstract

Depolarisation of ischaemic myocardial cells is at least partly due to loss of cellular potassium. Whereas most manifestations of ischaemia vary with heart rate potassium loss, however, reportedly does not. Cellular depolarisation was therefore correlated with extracellular potassium activity during serial coronary artery occlusions in Langendorff perfused canine hearts. Occlusions in sinus rhythm (92(11) beats X min-1) were alternated with rapidly paced occlusions (180 beats X min-1). For each occlusion cellular depolarisation was estimated from TQ depression and compared with the simultaneous increase in potassium electrode potential, delta EK. Although potassium accumulation accounted for most of the estimated depolarisation at slow heart rates, a potassium independent mechanism predominated during rapid pacing. The potassium independent mechanism was especially important in the first minute of ischaemia when pacing increased depolarisation by 324%, with little increase in delta EK. It appears that ischaemia induces a rate sensitive depolarising membrane current, which worsens conduction and promotes arrhythmias.

Abstract

Although lanthanum ions (La+++) block calcium influx in cardiac cells, they may paradoxically accentuate the sodium-free contracture. We have therefore studied the effects of La+++ on the zero sodium response in chick embryonic myocardial cell aggregates. Zero sodium alone causes: (a) A maintained contracture; (b) Asynchronous localized contractions that are selectively inhibited by caffeine or ryanodine, and presumably reflect release of calcium from the sarcoplasmic reticulum; (c) A nonspecific conductance increase that is ascribable to calcium-activated ion channels. Addition of La+++ potentiates the sodium-free contracture, and causes similar potentiation of the localized contractions and the conductance increase. All three phenomena occur 5-10-fold faster in 1 mM La+++ than in sodium-free fluid alone. In contrast, when La+++ is combined with caffeine or ryanodine, the zero sodium response is suppressed. We conclude that the paradoxical effect of La+++ on the contracture is not due to calcium influx, but to enhancement, or disinhibition of intracellular calcium release. Relaxation of normal myocardium may involve control of spontaneous calcium release by lanthanum- and sodium-sensitive calcium transport across the surface membrane.

Abstract

Calcium channel blockers suppress early ischemic arrhythmias, possibly by diminishing intracellular calcium overload and its effect on the ventricular action potential. To explore this, we compared the effects of diltiazem on ischemic "injury" potentials and ventricular fibrillation during serial coronary artery occlusions in dogs. Injury potentials and ventricular fibrillation were elicited every 15-25 minutes by simultaneous occlusion of the left anterior descending and circumflex arteries during rapid atrial pacing. DC epicardial electrograms were recorded differentially between the ischemic region and a small nonischemic region supplied by a proximal branch of the left anterior descending artery. Injury potentials developed with a uniform time course during five control occlusions, but were reduced by diltiazem infusion (0.5 mg/kg over 25 minutes) in each of eight dogs. The mean diastolic injury potential (T-Q depression) at 150 seconds of ischemia was 9.1 +/- 2.7 mV before diltiazem and 6.1 +/- 1.6 mV afterward (P less than 0.001). Diltiazem increased the mean time between coronary occlusion and ventricular fibrillation from 186 to 366 seconds (P less than 10(-5), but did not change the magnitude of the diastolic injury potential at onset of ventricular fibrillation. Diltiazem also delayed ischemia-induced conduction impairment to the same extent that it delayed injury potential development. In five dogs, the effect of diltiazem on regional blood flow near the epicardial electrodes was measured by infusion of radionuclide-labeled microspheres. Coronary occlusion reduced flow to the ischemic zone from 0.86 to 0.05 ml/min per g (P = 0.001). Diltiazem increased preocclusion flow by 11% (P = 0.03), but did not significantly alter flow during occlusion. Hemodynamic measurements show that diltiazem did not diminish cardiac work. Diltiazem therefore produced a flow-independent reduction of cellular depolarization during ischemia, which may be due to relief of calcium overload, and which may explain the antifibrillatory effect.

Abstract

Cardiac automaticity is partly due to a diastolic sodium current. Possible mediators of this include tetrodotoxin-sensitive "fast" channels, cesium-sensitive time-dependent pacemaker current channels, calcium-gated nonspecific channels, and electrogenic sodium-calcium exchange. We have studied the effects of abrupt sodium removal on membrane current and conductance in voltage-clamped chick embryonic myocardial cell aggregates, in the presence of various sodium flux inhibitors. Total replacement of sodium by lithium, Tris, or tetraethylammonium ions in aggregates clamped in the pacemaker range caused a brief outward current followed by a sustained net inward current. The outward current reached a peak value of 1.1 +/- 0.5 microA/cm2 at a mean latency of 5.4 +/- 1.2 sec. (n = 6; V = -70.5 +/- 8.9 mV; Tris). Conductance often decreased during the outward current. The inward current developed exponentially (t = 19 +/- 5 sec) and reached a steady state value of -1.6 +/- 0.4 microA/cm2. This current was reversed by depolarization (mean reversal potential = -13 +/- 13 mV), and was accompanied by increased conductance and spontaneous mechanical activity. Neither of the sodium-removal currents was affected by 20 microM tetrodotoxin. Cesium (up to 20 mM) had no effect on the late inward current or the mechanical activity, but decreased the early outward current by 80 +/- 12%. Manganese (25 mM), which blocks sodium-calcium exchange, abolished the late inward current and the mechanical activity. Manganese also reduced the early outward current by 27 +/- 10%. Manganese and cesium together blocked all the effects of sodium removal. We conclude that removal of extracellular sodium interrupts a cesium-sensitive "background" current, that may be related to the time-dependent pacemaker current, If. Sodium removal also causes gradual activation of a nonspecific conductance, which can ultimately depolarize the cells, and which may be gated by cytoplasmic calcium.

Abstract

Electrical excitation of cardiac muscle may sometimes be due to initiation of inward current by the presence of Ca2+ ions at the inner surface of the cell membrane. During digitalis toxicity and other conditions that abnormally augment cellular Ca2+ stores, premature release of Ca2+ from the sarcoplasmic reticulum leads to a transient inward current, which is large enough to initiate premature beats and is accompanied by a transient contractile response. This inward current may be mediated either by electrogenic sodium-calcium exchange or by specific Ca2+-activated cation channels that have recently been characterized in tissue cultures of cardiac myocytes. An obvious question raised by these observations is whether release of the sequestered Ca2+ stores during each normal beat exerts a similar influence on membrane potential. To explore this, chick embryonic myocardial cell aggregates were voltage-clamped during abrupt exposure to caffeine, which is known to release Ca2+ from the sarcoplasmic reticulum. The speed of the perfusion system and the relative absence of diffusion barriers in the tissue-cultured cells allowed the effects of caffeine-induced Ca2+ release to be studied on a time scale comparable to that of a single normal beat. We report here that abrupt exposure of the cells to caffeine produced a transient inward current having similar features to that of digitalis toxicity, and which was both large enough and rapid enough to potentially contribute to the action potential.

MECHANISM BY WHICH METABOLIC-INHIBITORS DEPOLARIZE CULTURED CARDIAC-CELLSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESClusin, W. T.1983; 80 (12): 3865-3869

Abstract

To elucidate the means by which metabolic inhibition depolarizes cardiac cells, spontaneously beating chicken embryonic myocardial cell aggregates were voltage clamped during superfusion with 2,4-dinitrophenol and iodoacetic acid. In aggregates continuously clamped in the pacemaker potential range, abrupt exposure to these metabolic inhibitors produced a slow transient inward current. This inward current was not due to an alteration of the pacemaker current, IK2, because it could still be elicited after IK2 was abolished by Cs+ ions. The inward current was increased by hyperpolarization and decreased by depolarization. It became larger and more sustained if intermittent action potentials were allowed during exposure or if the aggregates were pretreated with either 10 mM Ca2+ or 2.7 microM acetylstrophanthidin. The inward current was suppressed by removal of extracellular Na+ or Ca2+. These observations suggest that early depolarization of cultured cardiac cells by metabolic inhibitors involves some of the same mechanisms as the transient inward current of digitalis toxicity--specifically, an effect of intracellular Ca2+ ions on membrane permeability. Similar phenomena could occur during other forms of metabolic inhibition such as myocardial ischemia.

Abstract

In predicting vulnerability to ventricular arrhythmias and their response to antiarrhythmic therapy, several factors and questions need to be considered, including the following. (1) Ventricular tachycardia (VT) can be induced in most patients who have recurrent sustained VT. (2) Because induced VT can often be pace terminated, termination by pacing should be attempted before cardioversion is applied. (3) Acutely effective antiarrhythmic agents are no longer being found for the majority of patients undergoing electrophysiologic-pharmacologic study in our series. (4) Specific VT-induction programs affect both the inducibility of VT and acute efficacy of drugs at electrophysiologic study; this point requires examination. (5) Arrhythmia-induction studies may not product high yield in patients with clinical ventricular fibrillation or unsustained VT. (6) It is as yet unclear whether some drugs, such as amiodarone, can be accurately evaluated by using arrhythmia-induction techniques. (7) Induction of the repetitive ventricular response by V2 stimulation is not a useful method for antiarrhythmic drug selection in patients with recurrent ventricular tachyarrhythmias. (8) Adequate data do not yet exist to identify either the ECG-monitoring technique or the arrhythmia-induction technique as the preferred method for selecting antiarrhythmic drugs for chronic therapy of patients with recurrent ventricular tachyarrhythmias. A study to compare the accuracies of the two techniques is both feasible and needed.

Abstract

Calcium influx blockers reportedly suppress ventricular arrhythmias during acute ischemia. We therefore studied the effects of diltiazem and reduced serum ionized calcium on ventricular fibrillation (VF) in a reversible ligation model. VF was produced at 15-minute intervals by simultaneous occlusion of the left anterior descending and circumflex arteries of 31 dogs. Time from coronary occlusion to onset of VF showed no significant variation during 15 consecutive trials in six dogs that received saline alone. Intravenous infusion of diltiazem (0.02 mg/kg per min) markedly delayed the onset of VF in each of 10 dogs (P less than 0.0001). Mean VF latency increased from 138 to 295 seconds during a 45-minute diltiazem infusion, declined exponentially when the infusion ceased, and was strongly correlated with serum diltiazem concentration (r = 0.96, P less than 10(-6)). In five dogs, hemodynamic measurements, including coronary venous blood flow, were performed during each occlusion. The increase in VF latency by diltiazem was not due to increased coronary flow during occlusion or to reduction of left ventricular (LV) mechanical work. In six dogs, mean serum ionized calcium, [Ca++], was reduced from 1.11 to 0.59 mM by infusion of sodium citrate. Citrate infusion increased mean VF latency from 155 to 243 seconds, and the increase observed in each dog was correlated (r = 0.84, P less than 10(-6)) with the reduction in [Ca++]. VF latency was unaffected by lidocaine in nine dogs. The antifibrillatory effect of diltiazem during global LV ischemia may be an electrophysiological phenomenon related to reduction of cellular calcium influx.

Abstract

Sotalol and propranolol are nonselective beta-adrenergic blocking agents. Sotalol at low concentration, unlike propranolol, prolongs the duration of the transmembrane action potential. In a double-blind study, the electrophysiologic effects of intravenous sotalol (0.30 or 0.60 mg/kg; n = 9) were compared with intravenous propranolol (0.15 or 0.20 mg/kg; n = 8) in 17 patients with use of bipolar suction electrodes in the right atrium and right ventricle to determine whether sotalol prolongs the monophasic action potential duration in man. After administration of sotalol, there were significant increases (paired t test) in the Q-T interval (p less than 0.001), right atrial effective refractory period (p less than 0.05), right ventricular effective refractory period (p less than 0.005), right atrial monophasic action potential duration at 90% repolarization (p less than 0.01), and right ventricular monophasic action potential duration at 90% repolarization (p less than 0.005). Prolongation of the monophasic action potential duration was dependent on plasma sotalol concentration. There were no significant changes in these variables after propranolol. The spontaneous cycle length and Wenckebach cycle length increased significantly in both groups, and the mean blood pressure decreased in both, although not significantly after propranolol. In summary, sotalol but not propranolol prolonged atrial and ventricular effective refractory periods and lengthened the monophasic action potential and the Q-T interval of human myocardium after intravenous infusion. The ability to acutely prolong repolarization at therapeutic plasma concentration is unique among known competitive beta-adrenergic receptor antagonists.

Abstract

Calcium ions mediate the adverse effects of myocardial ischemia and have been implicated in the genesis of arrhythmias. Calcium influx blocking drugs protect against early ventricular arrhythmias during experimental coronary occlusion, and recent studies suggest that this effect is at least partly due to inhibition of myocardial cell calcium influx. Most of the pharmacologic maneuvers used to simulate acute ischemic arrhythmias in vivo also produce intracellular calcium overload. Production of calcium overload in small myocardial cell clusters causes fibrillatory electrical and mechanical activity similar to that recorded from fibrillating hearts. Fibrillation in these cell clusters is mediated not by reentrant conduction, but by the same subcellular processes that give rise to depolarizing afterpotentials and abnormal automaticity. Agents favoring calcium influx, such as beta adrenergic agonists, accentuate these processes, while agents that depress calcium influx inhibit them. Although the relation of these experimental models to clinical ischemic arrhythmias has not been fully delineated, calcium influx blocking drugs may prove useful in reducing the incidence of sudden cardiac death.

Abstract

1. Simultaneous recordings of membrane potential and edge movement were obtained in spontaneously beating chick embryonic myocardial cell aggregates, which are known to behave as an isopotential syncytium.2. The time course of edge movement was similar in different aggregates, and in different regions of the same aggregate.3. Peak amplitude was increased by 10(-6)m-ouabain, and by rapid reduction of the external sodium concentration.4. Peak amplitude was decreased during single premature action potentials, but sustained rapid pacing produced an ascending staircase.5. Depolarizing current pulses increased both the amplitude and duration of the contraction, and caused potentiation of the next spontaneous beat. Edge displacement during a series of pulses was a monotonic function of membrane potential.6. Edge movement between action potentials (diastolic movement) was well fitted by an exponential with a mean time constant of 69 msec. Diastolic edge movement was due to a weak, slowly decaying contractile force, which was demonstrated in cells grown on a linear-elastic nylon bristle.7. The time course of diastolic edge movement remained constant, or nearly constant, during variations in peak amplitude that resulted from prematurity of the action potential, exposure to 10(-6)m-ouabain, spontaneous mechanical alternans, or prolongation of the action potential by current pulses.8. In contrast, reduction of the external sodium concentration produced marked, selective slowing of the diastolic edge movement. Similar slowing occurred during cooling and during staircase. Diastolic edge movement was selectively accelerated when the preceding interbeat interval was prolonged by a hyperpolarizing current pulse.9. The above observations are consistent with the hypothesis that edge displacement is a monotonic function of contractile force.10. The slow relaxation between action potentials probably reflects removal of intracellular calcium across the surface membrane in exchange for sodium. Changes in the rate of calcium removal may play a role in the regulation of contractility in this tissue.

CORRELATION BETWEEN RELAXATION AND AUTOMATICITY IN EMBRYONIC HEART CELL AGGREGATESPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESClusin, W. T.1980; 77 (1): 679-683

Abstract

Diastolic depolarization in cardiac muscle is due to a decline in potassium permeability that has been ascribed to removal of intracellular free calcium. A continued decline in tension during the pacemaker potential might therefore occur. In this study, contractile responses of chicken embryonic heart cell aggregates are recorded with a photodiode. Photodiode output is well correlated with the position of the aggregate's edge. Movements of different edges are synchronous, and their amplitude and duration vary appropriately during experimental maneuvers that alter the magnitude and duration of contractile force. Edge movement during relaxation has two phases, a rapid phase lasting about 100 msec and a slow phase that may last over 10 sec. The slow phase is not due to viscoelasticity because its time course does not depend on the magnitude or duration of the initial deformation. The rate of relaxation is correlated with the rate of depolarization during the pacemaker potential. Reduction in automaticity during cooling, spontaneous variation, and overdrive pacing are associated with impairment of the slow component of relaxation. Electrophysiological evidence suggests that the diastolic potassium permeability of the aggregates is controlled by intracellular calcium. A possible explanation for the correlation between the slope of the pacemaker potential and the slow component of relaxation is that both phenomena reflect a common physiological process-i.e., the removal of free calcium from the cytoplasm.

Abstract

Tonic nerve activity in skate electroreceptors is thought to result from spontaneous activity of the lumenal membranes of the receptor cells which is modulated by applied stimuli. When physiological conditions are simulated in vitro, the receptor epithelium produces a current which flows inward across the lumenal surface. This epithelial current exhibits small spontaneous sinusoidal fluctuations about the mean that are associated with corresponding but delayed fluctuations in postsynaptic response. Small voltage stimuli produce damped oscillations in the epithelial current similar in time-course to the spontaneous fluctuations. For lumen-negative, excitatory stimuli, these responses are predominantly an increase over the mean inward current. For inhibitory stimuli they are predominantly a decrease. Increased inward current across the lumenal membranes of the receptor cells increases depolarization of the presynaptic membranes in the basal faces leading to increased release of transmitter and an excitatory postsynaptic response. Decreased inward current decreases depolarization of the presynaptic membranes leading to a reduction in transmitter release and an inhibitory postsynaptic response. Clear changes in postsynaptic response are detectable during stimuli as small as 5 microV with saturation occurring at +/- 400 microV. The evoked oscillations in epithelial current are damped and the postsynaptic responses decline during maintained stimuli with large off-responses occurring at stimulus termination. The initial peak of the off-response is similar to the response produced by onset of an oppositely directed stimulus. These observations substantiate the role of receptor cell excitability in the detection of small voltage changes.

Abstract

When physiological conditions are simulated, skate electroreceptors produce small maintained oscillatory currents. Larger damped oscillations of similar time-course are observed in voltage clamp. Subtraction of leakage in voltage clamp data shows that the oscillations involve no net outward current across the lumenal surface of the epithelium. The oscillations are much faster than the late outward current generated by the lumenal membranes of the receptor cells. Treatment of the basal surface of the epithelium with tetraethyl ammonium (TEA), high K, Co, or EGTA reversibly blocks the oscillations in voltage clamp, but has little or no effect on the epithelial action potential in current clamp or on the current-voltage relation. The TEA sensitivity of the oscillations indicates that they involve a potassium conductance in the basal membranes of the receptor cells. Treatment of the basal membranes with TEA and high calcium, with strontium, or with barium causes these membranes to produce large regenerative responses. Direct stimulation of the basal membranes then elicits a lumen-positive action potential whereas stimulation of the lumenal membranes elicits a diphasic action potential. Excitability of the basal membranes is abolished by extracellular Co, Mn, or La. Modulation of the lumenal membrane calcium conductance by the basal membrane conductances probably gives rise to the oscillatory receptor currents evoked by small voltage stimuli. The slower calcium-activated late conductance in the lumenal membranes may be involved in sensory accommodation.